During the past decades immunologists have deciphered many of the mechanisms by which the mammalian immune system either combats or mediates a host of diseases, and clinicians are beginning to translate these insights into novel immunotherapies. However, current technologies are often inadequate to promote further progress in our understanding of cellular and molecular immune pathways, and the available tools to 'engineer' immune cells to harness their full therapeutic potential are very limited. One major barrier is the lack of effective and robust intracellular delivery methods that could enable investigators to directly probe and manipulate intracellular pathways in primary immune cells. Modulating immune cell function through intracellular delivery of biomolecules has many potential applications. Delivery of macromolecules, such as polysaccharides, proteins, nucleic acids or multi-component nanostructures, to the cell cytoplasm can transiently or permanently alter cell function for research or therapeutic purposes. Herein, we propose to develop a vector-free microfluidic platform to deliver macromolecules and other payloads with high efficiency directly into the cytosol of primary mouse and human immune cells. The principle underlying this approach is temporary membrane disruption by rapid mechanical deformation, or squeezing, of immune cells to facilitate uptake of loading material in the fluid medium. Our preliminary data indicate that the proposed mechanical membrane disruption approach to intracellular delivery could potentially overcome the challenges encountered by traditional technologies, such as vector-based or electroporation-mediated systems, and enable delivery of a diversity of materials to manipulate cell function. Indeed, preliminary comparisons to alternative delivery methods demonstrate that our platform is capable of delivering functional protein and siRNA with equal or greater efficiency, while showing fewer off-target effects and maintaining genome integrity. We hypothesize that the proposed microfluidic cell squeezing platform can be harnessed to control immune cell fate and function. The key objectives of this project are to: i) generate an optimized library of microfluidic chips for use with a panel of relevant immune cell subsets, ii) characterize potential side-effects of the delivery system, and iii) use the novel capabilities of this technology to address the challenge of protein delivery. If successful, this work would facilitate the commercial launch of an enabling new technology platform and generate the preliminary data necessary to justify future work on platforms for ex vivo engineering of immune cells in therapeutic applications.